Receptor tyrosine phosphatases (RPTPs) play important roles during neurogenesis, cell proliferation, and ne ronal positioning in the nervous system, but their mechanisms of action remain poorly understood. Genetic analyses have been complicated by the overlapping functions of different RPTP subtypes1, 2, while authentic ligands for many RPTPs remain unknown3. Recent studies using the model system Manduca sexta have identified a novel function for a specific RPTP that may clarify these issues. In the developing enteric nervous sy tem (ENS) of Manduca, placode-derived neuroblasts must migrate extensively before differentiating into ma- ture neurons, during which they travel on pre-formed muscle band pathways while avoiding the enteric midline. Previous work showed that the midline muscles express the sole Eph receptor tyrosine kinase in this system (MsEph), while the neuroblasts express its cognate Ephrin ligand (MsEphrin; a GPI-linked or type-A Ephrin)4. Intriguingly, reverse signaling via MsEphrin prevents the neuroblasts from crossing the MsEph-expressing midline cells, a response that involves the local activation of a Src family kinase (SFK) and retraction of their leading processes5, 6. In contrast, conventional forward signaling via MsEph receptors plays no role in this process, providing the first example of Ephrin-A reverse signaling in the control of neuroblast positioning. However, the mechanisms by which GPI-linked Ephrins transduce signals across the membrane remain poorly understood. An affinity screen for MsEphrin co-receptors identified PTP10D, a type-III RPTP that regulates midline axonal responses in the CNS2. Preliminary studies showed that PTP10D is co-expressed with MsEph- rin by the neuroblasts, while inhibiting PTP10D expression induced the same pattern of ectopic midline cros- sovers caused by blocking MsEphrin signaling. Intriguingly, the PDZ adapter protein ZO-1 (zonula occludens- 1) was also identified in this screen, and shown to co-localize with MsEphrin and PTP10D in the neuroblasts. Originally classified as a tight junction protein7, ZO-1 can also regulate cell migration in vitro8, 9 and may interact with RPTPs and SFKs in a variety of contexts10-12. The overall objective of this proposal is to determine how PTP10D regulates neuroblast migration in the developing ENS. The central hypothesis is that PTP10D acts as a co-receptor for MsEphrin, coupling this GPI-linked Ephrin with ZO-1 and its downstream effectors. Specific aims will test the role of PTP10D and ZO-1 in MsEphrin-dependent aspects of migration, using published methods to manipulate gene expression and protein interactions in embryo culture. These studies will provide essential new data for an R01 application, with the goal of comprehensively defining the mechanisms of Ephrin-A reverse signaling in the nervous system. Public Heath Relevance: Demonstrating novel roles for RPTPs and ZO-1 in Ephrin-A signaling will provide new tools for investigating why human mutations that affect Ephrin-As result in defective brain growth13, and for developing new therapeutic strategies that address neuro- degenerative conditions in which Ephrin-A reverse signaling may be misregulated14-16. PUBLIC HEALTH RELEVANCE: The proposed research will investigate a novel role for receptor tyrosine phosphatases (RPTPs) as co- receptors for type-A Ephrins in the control of neuroblast migration. RPTPs and Ephrin-As have been independently linked with normal brain development and neurogenic responses to traumatic injury, but their functional interactions with the adapter protein ZO1 have not previously been recognized. Defining the mechanisms by which RPTPs and ZO-1 transduce Ephrin A-dependent signals in a model system will provide a new perspective on how this signaling pathway may regulate neuronal differentiation in both the embryonic an adult nervous system, and in the context of neurological disorders in which RPTP-Ephrin interactions may be impaired.